Antigen for determination of syphilis and process of making the same from yeast



May l2 1953 P. FUGAzzoTTo 2 638 4 l ANTIGEN RoR DETERMINATION oF sYPHILIs AND 35 PROCESS oF MAKNG THE SAME FROM YEAsT yFiled Nov. 17, 1948 INVENTOR. PMI/z Fas-azz afro.

ALJ/@JM Patented May 12, 1953 Arrivierent Eon; narnnmnn'nonors-Yeni- Lrs.; AND. PRoeEss; on MAKING 'me SAME PaulnFugazzotto; Indianapglisplnd. Application Noyembeeiv, 194s;y sexier No. Awitze f sentirne.. (el. reife-.8425

i This invention relates to a substance' common-- 1y known as an antigen. and to the proce-ss' of" making the samer Morespeciiically, the invention relates to an antigen or indicator for the detern-lination or the. presence,A ot syphilis..

Heretofore, there. have been numerous. pro.- cedures proposedzfor thev production, ot antigens, together with the processes of making. them.. These antigens have been, used .with varionsit'echniquesEk for testing body' uid's. forV evidence.. oi" syphilis..v While improxzementsihave. been made.

inthe quali'tpof the, antigens.employed,V all' have.

heretofore been found lacking inthat nonspeciie reaetions have ,attendedtheir use., Furthermore, eacii of;` thesev antigens. hasy left. much, to bie dief sired'. in providing.moreisensitive results inthe' seraof syphilitiepatients..

. In the antigens heretoforeemployed, the basic rawmaterial usedhasbelenof animal'ori'gin; en, beef` heart; or.. from beef. heart rplus egg yolk.: for example;l even the popularzednardiolipin antigen has` been. obtained from beefheart;

Itisthe, primary obj ect' ojthe' present invention to'. provide, an. antigen which.possessesthequalityoay high.V degree; of specificityv combined with" a high. deg'reeof sensitivity:

The primary feature of the present invention resides; in the use of an antigen derived frorn'a raw materiarofvegetable' origin, and more. par-vv ticnlarl'y yeast;

It is a furtherA @bientotthepresent invention toprovide an antigen. possessing. comparable physical and serological properties to those antigensprepared from .raw material of animal origin such a-sbeef 'heart `or beef heart plus eggyol-k.

It' is a stilliui't'her object' of'the present inventiofrr ta provide' arr antigen- 01E`v sue-h character that uniform resultsfwill attend its use.

It has been determined that antigens formed from raw materials of animalforigi'n are. frequently unpredictable. For example; great care must be exercised in; handling heef heartv from the moment it i's removed from-the animalunti-l the dehydratectpulverized musclehas been. subfjected to the Avarious extractions.- No sini-rieA manipulation oregroup-of manipulationahasheretoffore been devised toenable the usertei-predict` with any'degreeof certaintyv of the. success ofA the` antigen to be prepared therefrom;

It isa still further objectof the present iiiven-- tion to produce an antigen' which may beV pre duced Without elaborate or expensive purification procedures and which will result in a uniform antigen. f

The antigen Tnefraw material.. insect' as; a source omnia newV antigen` yeastobtainable. on. the onen market 2@ best results have beensobtainectf witlrhaliersiyeaste; whieh or courseV can bei cuituredi and: propagated; onf ai suitable medium; irri. the.- laboratory, its. been*Vv determinada than brewer s yeastgalsei container the-antigeen ingradient;Y

Likeibeeiifheamt antigens. employed at theipreSi-f. entitinfre;A extraciisf, ai". yeast?. are eh'iefdyf llQOdai: compesitiong. although,investigation; @ii Qompar tiyeiextrantions; indicates they Wesens@ offL ai p ,p ticipating carbohydratecomponentaiiparelifl S a. side.: chain ini a,- lipixif; molecule; extrae are. alsoisimi'lar teQ beef; heart.. a tige ten'y orf-t physio; thesyeaisti eells te tien.r the limiecle:1

suela; yeasty eatraetsxdier therefrem; arefm h purer 'Ehat2is,-.theygaraleSSinol ed` `r sented onfliet-ne nenfnartcipatng or I-a1se--v reacting? @o1-.neonerits-r Preiminary' preparaft'i'on 07T` raw' materia? y' Wfiiereir anti-ve bakersf yeast is; lobtained.in.; the dehydrated states. it is ground-.inta a. ratherl powders throughthe useof: sterile: clean` dry. sanai or anyz other; convenient@ means. Where: actie/le. bakers.yeastrobtained; in the moist state feit-.tirar` cnthei-openrmarket or; from a; laberatoryz culture' mediumr iausediii: shonldizsti be, dehydrated: follower.

Iespread; out dishes glas r11 cwba'fm.: heidi` at:

.ing process. thereby assuring ai piner. antigen...

oughl-y for aboutten. minutes, preferably W h a mechanical mixer, and the. Wah Water then moved. einer. by maaien 1n a.. Buche under reduced'preSSure, or better stilljbyY centr ugation. The Washing isrepeated. twoj. Qrmore:

times in the same manner, exceptthat vernlinutes of thorough stirring is desirable before removal of the wash water. After the material is Washed suiciently, it is subjected to the same drying process as described above in connection with active bakers yeast obtained in the moist state.

Preparation of antigen extract The extraction of the yeast powder is accomplished as follows:

An amount of powder is weighed and then transferred to a mortar or a Waring Blendor to which is added just enough 95% ethyl alcohol to make a rather thick fluid paste. The mixture is ground for 15 minutes if it has been placed in a mortar or for five minutes if a Waring BlendorV is used. During the grinding operation small portions of alcohol are added to maintain the thick fluid consistency of the material. The mixture is then transferred to a Pyrex bottle of suitable size, which is stoppered with a glass cork or tin foil covered cork; a volume of 95% alcohol equivalent to 4.0 ml. for each gram (original dry weight of powder) is then added. Some of this alcohol should be used to aid in transferring the material to the Pyrex bottles. The extracting mixture is placed in the dark at room temperature and agitated thoroughly several times a day for two days. After it has stood overnight undisturbed the supernatant liquid is decanted into suitable centrifuge tubes (or a filter) to be cleared. The clear extract is stored in appropriate Pyrex bottles and the residue, if any appreciable amount was carried over from the extracting bottle, is returned thereto. To all of this residue is added 2.0 ml. of 95% ethyl alcohol per gram (original dry weight) of yeast employed, and a second extraction is made as above. The supernatant liquid is decanted as in the first extraction and then a third extraction is made as in the second extraction.

Finally, the extracted residue is washed three times with a volume of 95% alcohol equivalent to 1.0 ml. for each gram (original dry weight) of yeast employed, sedimenting the residue each time in the centrifuge at about 2000 R. P. M. to obtain the clear supernatant. All washings and extractions are pooled in a Pyrex bottle and agitated several times daily for three or four days. Approximately one-third of the extract is set aside while cholesterol (Phanstiehl ash-free) is added to the major portion in an amount equivalent to 0.5 gram per 100.0 ml. of extract. The bottle of extract may be heated in a water bath at 56Q C. to hasten solution of the cholesterol, and the final product is then filtered (gravity filter) before standardization.

Use of the antigen In order to use this cholesterolized extract of yeast in the serology of syphilis, it is necessary first of all to prepare a suitable suspension of the dissolved lipoids. This is accomplished by adm ixture with physiologic salt solution (amount determined by titration). The customary methd of making this mixture is (as described by Kolmer for beef heart antigens) by adding the extract drop by drop to the required amount of saline. However, it has been found that more sensitive suspensions are obtained if the extract is first mixed with a small amount ofsaline (as determined by experimentation) and allowed to stand from seven to ten minutes before the mixture is brought up to proper dilution with further addition of saline.

4 The test To tubes containing 0.02 ml. of inactivated patients serum (or 0.1 to 0.2 ml. of spinal iluid) is added 0.1 ml. of the antigen suspension mentioned above. The tubes are thoroughly shaken and allowed to stand at room temperature for from ten to twenty minutes before the required amount of guinea pig complement is added (usually two units in diagnostic tests). The tubes are again agitated, placed in a refrigerator at from four degrees to eight degrees C. for from four and one-half to five hours, and then at room temperature for fifteen minutes. Sensitized red blood cells (sheep) are added-0.2 ml. of 21/2% suspensionthe tubes agitated and then placed in a water bath at 37 C. for fifteen minutes. The final results are read and recorded in the customary manner.

Comparative tests A properly standardized antigen is one which possesses a high capacity for detection of the presence of syphilis. However, it is likewise desirable that the antigen avoid false reactions in conditions other than syphilis. All antigens developed to date by the various authors have been known to give certain percentages of false reactions. This includes even the highly puried cardiolipin antigens.

Tests have been made with the present antigen through the medium of the complement fixation testing system using guinea pig serum. As is commonly known, this serum is one of the reagents used in this type of test to aid in the detection of syphilis in a patients sera. Any antigen which reacts with the guinea pig (complement) sera will give a tendency towards false reactions when employed in routine tests on human sera and spinal fluids'. In the following table are given results of complement fixation tests for syphilis on a group of 27 guinea pigs tested with the yeast antigen of this invention as compared to four other well known antigens. The guinea pig numbers that are missing represent those which were negative with all antigens:

SPECIFICITY COMPARISON No'rE.-The N. Y. and Harris antigens are cardiolipin antigens. The KoL represents theKolmer complement fixation antigen. The Kahn 162 antigen is the Kahn flocculation test antigen titrated for use 1n the complement fixation tests. All antigens were used at opt1mal dilution under identical conditions. The number and symbols represent degrees of hemolysis from zero (00 tov40, 50, 60, 70, et cetera) to complete, representing highly positive (00) to weaker positive (40, 50, 60, et cetera) to negative respectively.

From this table it is evident that the ycardiolipin antigens showing. a great percentage of negatives would be less likely to give false reactions when employed in routine tests. In 22 out of the 27 guinea pig tests there Was a reaction with the routine Kolmer antigen `and with the particular lot (162) of -the Kahn antigen. Only 2 of the 27 reacted with the yeast antigen indicating that this antigen is less likely to give false reactions than even the cardiolipin antigens which reacted with four guinea pigs in one series of tests (the N. Y.) and in five with the other (Harris). It is, therefore, evident that the yeast antigen does meet the requirement of minimal reactivity with guinea pig serum, which as has already been stated is used as a reagent in complement fixation tests.

Experience With many other antigens, particularly those extracted from beef heart, has shown that failure of an antigen to react with guinea pig serum, though extremely essential, does not necessarily mark it as a superior product, for such other antigens may also be undersensitive to syphilitic sera as well. With this experience in mind, comparative tests were made on human specimens to check the yeast antigen for this property of sensitivity. The following chart shows the results:

SENSITIVITY COMPARISON Antigen Serum Specimen No.

Klgn Yeast NorEf-The results given (T, i, 1, 2, 3, 4+).represent, respectively, increasing order of positivity from negative to strong positive (4).

It is apparent from these tests that the yeast antigen not only is more specific in that it displays a lower reactivity with false reacting sera (guinea pig), but as Well, is just as sensitive as the beef heart antigens currently in use.

Comparative tests were also made on a group of 116 patients sera of whom 35 were known syphilitics and 81 were known to be nonsyphilitic. Of the syphilitics all but 4 were found to react with the yeast antigen. Of the nonsyphilitics only two gave reactions in the diagnostic tests, but these Were not strong enough to be reportable. These 116 specimens were part of a series of approximately 350 specimens distributed by the United `States Public Health Service in the annual serology evaluations of 1948.

In the following table are given the comparative results in connection with the above mentioned tests, of the syphilitic specimens for which the United States APublic Health` Service (V- series 1948v evaluation survey:

Antigen T Kolmer- Yeast- Spemmen ho' Tube Nos. Tube Nos.

2 3 3 4 4 Neural Syphilis. 2 4 4 Do. 2 3 4 4 D0. Do. Early Latent Syphilis.

3 4 4 .4 i D0. x 3 4 3 4 4 Neural Syphilis. 4 Late Latent Syphilis. x l x 3 Primary Syphilis. :l: 3 4 Late Latent Syphilis.

2 3 4 4 4 Do. :l: 4 4 Cardiovascular Syphilis.

2 3 4 4 4 Neuro Syphilis. 3 3 4 3 4 4 Visceral Syphilis. Late Latent Syphilis. Late Latent Primary Syphilis.

In conclusion, it is apparent that the antigens prepared from yeast, as heretofore described, have been found to offer minimal interference in the presence of reagents (guinea pig serum) used, and to compare more than favorably With other antigens in complement fixation tests on non-syphilitic sera. This present antigen appears to have a sensitivity equal to or higher than other antigens already in use and with smaller risks of giving false reactions. It has not been necessary in the production of this antigen to resort Ito the rather elaborate procedures described in the literature of other authors for their antigens, involving as they do preliminary extractions with ether, acetone, or both; precipitations; evaporations and redissolutions, all of which are ltime consuming, wasteful and in every way expensive, especially since these manipulations are merely preparatory in most cases to the nal extraction with alcohol. With the exception of the simple washing which appears to be necessary in the case of brewers yeast and the dehydration of the yeast in the moist state, no other manipulation appears to be required preparatory to the alcohol extraction of the desired component from the yeast.

While the invention has been described in great detail in the vforegoing description, such detail is to be considered as illustrative only and not restrictive in character.

The invention claimed is:

1. An antigen for use in serologic tests for syphilis, comprising yeast lipoids derived from extraction of dehydrated yeast with alcohol, with cholesterol added thereto.

2. A diagnostic antigen for serologic testing, comprising substances derivable from dehydrated yeast by extraction with ethanol at room temperature, to which cholesterol has been added.

3. An antigen for use in serologic tests for syphilis consisting of an ethanol solution containing cholesterol and water-insoluble lipoids derived from dehydrated yeast.

4. The process for the preparation of antigenic material from yeast, which comprises extracting commnuted dehydrated yeast with substantially Water-free alcohol at room temperature, removing undissolved material from the alcoholic extract, and adding cholesterol to the resulting alcohol solution.

5. The process according to claim 4, in which the alcohol is 95% ethanol.

PAUL F'UGAZZOTTO.

8 References Cited in the le of this patent UNITED STATES PATENTS OTHER REFERENCES J. Biol. Chem., volume 137, February 1941 pages 525 to 533. 

1. AN ANTIGEN FOR USE IN SEROLOGIC TESTS FOR SYPHILIS, COMPRISING YEAST LIPOIDS DERIVED FROM EXTRACTION OF DEHYDRATED YEAST WITH ALCOHOL, WITH CHOLESTEROL ADDED THERETO. 